1. Field of the Invention
This invention relates to novel alkaline cellulases and also to microorganisms which are able to produce the same, belong to the genus Bacillus, and grow up in a neutral medium.
2. Description of the Prior Art
The development of cellulases, which are cellulose-decomposing enzymes, has been made for the purpose of effectively utilizing biomass resources and particularly, cellulose resources. A diversity of strains have been isolated as cellulase-producing fungi or bacteria including, for example, not only molds of the genera Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Acremonium and the like, but also bacteria of the genera Pseudomonas, Cellulomonas, Ruminococcus, Bacillus and the like and actinomycetes of the genera Streptomyces, Thermoactinomyces and the like. At present, however, cellulases for biomass have not been frequently utilized on an industrial scale.
On the other hand, studies have been made on novel industrial utility of cellulases as an ingredient for detergents for clothes, to which attention has now been paid (Japanese Patent Publication Nos. 59-49279, 60-23158 and 60-36240). Most cellulases produced by microorganisms in the natural fields are classified as so-called neutral or acidic cellulases which exhibit optimum and stable enzymatic activity in a neutral to acidic range. Only a few cellulases are so-called alkaline cellulases which meet the requirements for formulation in detergent compositions for clothes or can exhibit an maximum activity in an alkaline pH range, and so-called alkali-resistant cellulases which have an alkali resistance. The term "alkaline cellulase" used herein is intended to mean one whose optimum pH is in an alkaline range, and the term "alkali-resistant cellulase" means one whose optimum pH is in a neutral or an acidic range, but which has a satisfactory activity as compared with an activity at an optimum pH and is maintained stable in an alkaline range. The term "neutral" means a pH range of from 6 to 8, and the term "alkaline" means a higher pH range.
For the production of alkaline cellulases and alkali-resistant cellulases usable in detergent compositions for clothes, only several methods have been proposed. These methods include, for example, a method of collecting cellulase A by cultivation of alkalophilic bacilli belonging to the genus Bacillus (Japanese Patent Publication No. 50-28515), a method of producing alkaline cellulase 301-A by cultivation of alkalophilic bacteria belonging to the genus Cellulomonas (Japanese Patent Application Laid-open No. 58-224686), a method of producing carboxymethyl cellulase by cultivation of alkalophilic Bacillus No. 1139 (Fukumori, F., Kudo T. and Horikoshi, K., J. Gen. Microbiol., 131, 3339, (1985)), and a method of producing an alkaline cellulase by the use of one strain belonging to the genus Streptomyces (Japanese Patent Application Laid-open No. 61-19483). However, these methods are all unsuitable for the industrial fermentation production.
In recent years, we have found that Bacillus sp. KSM-635 (FERM P-8872), which is one of alkalophilic bacteria, can efficiently product alkaline cellulase K which is suitable as an ingredient for detergents for clothes and that proper selection of cultivation conditions enables one to enhance the productivity and conduct industrial fermentation production of the alkaline cellulase.
However, the cultivation conditions of the Bacillus sp. KSM-635 are not always advantageous from an industrial point of view. More particularly, an alkalophilic strain should be cultivated under alkaline pH conditions during the cultivation. A so-called alkaline fermentation process using alkalophilic strains has just been started, and a full knowledge on the physiological and biochemical properties of these alkalophilic microorganisms has not been obtained. Thus, difficulties have been involved in the preparation of media and the manner of cultivation sufficient to effect the industrial production by fermentation.
Moreover, the true alkaline cellulases of the afore-described documents which have an optimum pH in an alkaline region, are enzymes which are produced by Bacillus N1 strain, N2 strain and N3 strain (Japanese Patent Publication No. 50-28515) and have optimum pHs of 8 to 9, 9 and 8 to 9, respectively, an enzyme produced by Bacillus No. 1139 and having an optimum pH of 9, and alkaline cellulase K produced by Bacillus sp. KSM-635 and having an optimum pH of 10 (Japanese Patent Application No. 61-257776). Now, there is a demand for alkaline cellulase which have an optimum pH in an alkaline region and can be suitably formulated in detergent compositions and which have a wide working pH range.